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Influenza A virus poses significant public health challenges due to its high mutation rate and zoonotic potential. Whole-genome sequencing (WGS) is crucial for monitoring and characterizing these viruses. Oxford Nanopore Technologies (ONT) and Illumina next-generation sequencing platforms are commonly used, with ONT being advantageous for its long-read capabilities, portability, and unique ability to access raw data in real-time during sequencing, making it suitable for rapid outbreak responses. This study optimizes the ONT Ligation Sequencing Influenza A Whole Genome protocol by refining RT-PCR kits, primers, and purification methods, and evaluating automation for high-throughput processing. The alternative RT-PCR kits, combined with alternative primers, significantly improved read depth coverage and reduced short, untargeted reads compared to the original ONT protocol. The improvement was particularly evident in the minimum read depth coverage of polymerase segments, which often face challenges with achieving uniform coverage, displaying higher coverage at the 5' and 3' termini, and lower coverage in the central regions. This optimized protocol for targeted influenza A WGS not only enhances sequencing quality and efficiency, but is applicable to all NGS platforms, making it highly valuable for studying influenza adaptation and improving surveillance. Additionally, this protocol can be further refined and adapted for the sequencing of other pathogens, broadening its utility in various pathogen monitoring and response efforts. [ABSTRACT FROM AUTHOR]

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time‐consuming. Using a reverse workflow for specimen sorting and identification leveraging high‐throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology‐based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity. [ABSTRACT FROM AUTHOR]

Oxford Nanopore Technology (ONT) allows for the rapid profiling of aquaculture microbiomes. However, not all the experimental and downstream methodological possibilities have been benchmarked. Here, we aimed to offer novel insights into the use of different library preparation methods (standard-RAP and native barcoding-LIG), primers (V3–V4, V1–V3, and V1–V9), and basecalling models (fast-FAST, high-HAC, and super-accuracy-SUP) implemented in ONT to elucidate the microbiota associated with the aquatic environment and farmed fish, including faeces, skin, and intestinal mucus. Microbial DNA from water and faeces samples could be amplified regardless of the library–primer strategy, but only with LIG and V1–V3/V1–V9 primers in the case of skin and intestine mucus. Low taxonomic assignment levels were favoured by the use of full-length V1–V9 primers, though in silico hybridisation revealed a lower number of potential matching sequences in the SILVA database, especially evident with the increase in Actinobacteriota in real datasets. SUP execution allowed for a higher median Phred quality (24) than FAST (11) and HAC (17), but its execution time (6–8 h) was higher in comparison to the other models (0.6–7 h). Altogether, we optimised the use of ONT for water- and fish-related microbial analyses, validating, for the first time, the use of the LIG strategy. We consider that LIG–V1–V9-HAC is the optimal time/cost-effective option to amplify the microbial DNA from environmental samples. However, the use of V1–V3 could help to maximise the dataset microbiome diversity, representing an alternative when long amplicon sequences become compromised by microbial DNA quality and/or high host DNA loads interfere with the PCR amplification/sequencing procedures, especially in the case of gut mucus. [ABSTRACT FROM AUTHOR]

AbstractWe sequenced and assembled mitochondrial genomes of three tenthredinid sawflies (Euura poecilonota, E. striata, and Dolerus timidus) using Oxford Nanopore Technologies’ MinION. The Canu assembler produced circular assemblies (23,000–40,000 bp). Still, errors were found in the highly repetitive non-coding control region because of the fragmented DNA which led to no reads spanning the complete control region, preventing its reliable assembly. Based on the non-repetitive coding region’s sequencing coverage, we estimate the lengths of mitochondrial genomes of E. poecilonota, D. timidus, and E. striata to be about 30,000 bp, 31,000 bp, and 37,000 bp and control region to be 15,000 bp, 16,000 bp, and 22,000 bp respectively. All standard bilaterian mitochondrial genes are in the same order and orientation, except trnQ, which is on the minus strand in Euura and the plus strand in Dolerus. Using published tenthredinid genome data, we show that control region lengths are often underestimated. [ABSTRACT FROM AUTHOR]

Species extinctions increase at a global scale; therefore, rapid inventorying of our planet's biodiversity is becoming more and more important. As insects represent the highest portion of the fauna and play key ecological roles, it is a pressing need to investigate their biodiversity and accelerate species discovery, especially for understudied insect groups, also known as "dark taxa." Phoridae (Diptera) are a great example of a "dark taxon," in particular the genus Megaselia Rondani.The use of integrative methodologies is the best approach to face up to the task of describing hyperdiverse and dark taxa, as morphology alone can be imprecise and slow, and molecular methods alone are often insufficient and lead to errors.Here, we used the Large‐Scale Integrative Taxonomy (LIT) approach to sort 9000 Megaselia into 277 putative species based on DNA barcodes. Each cluster passed through an evaluation of the predictors for incongruence indices between clusters and morphology (maximum p‐distance, stability index), and a subset of specimens were morphologically examined.We provided species estimates with Chao1, and our results suggest a 15% increase in species richness on our dataset. As this estimate was mostly based on samples from southern Germany, the species count will likely increase with expanded geographic sampling. This is a step forward in the study of this taxon, despite the fact that the German insect fauna is one of the best known in Europe and boasts more than one hundred years of study on phorids. [ABSTRACT FROM AUTHOR]

Several Xiphinema nematode species are vectors of regulated nepoviruses. The aim of this study was to develop an optimized method to detect virus-carrying Xiphinema specimens. The study compared various techniques for nematode extraction from soil (automated zonal centrifuging and the Flegg modified Cobb method), nematode cuticle disruption (slicing, bead beating, and bead beating with collagenase), RNA extraction (RNeasy Plant Mini Kit, cetyltrimethylammonium bromide [CTAB], and KingFisher MagMAX RNA Isolation Kit), and nepovirus detection (nepovirus generic subgroup and species-specific quantitative and conventional reverse transcription polymerase chain reaction [RT-(q)PCR] assays, as well as shotgun sequencing with Oxford nanopore sequencing technology using a MinION device). Based on this comparison, a diagnostic procedure is proposed including the best-performing methods: nematode extraction using automated zonal centrifugation, physical disruption of nematode cuticle using bead beating or slicing, and RNA extraction using either the KingFisher MagMAX or the RNeasy Plant Mini Kit method. While existing generic nepovirus subgroup reverse transcription polymerase chain reaction (RT-PCR) assays lacked sensitivity, species-specific RT-(q)PCR assays successfully detected Arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), and tomato ringspot virus (ToRSV) in X. diversicaudatum, X. index, and X. americanum sensu stricto (s.s.), respectively. The reliability of nepovirus detection was higher with adults compared with juveniles. The minimum nematode quantity required varied depending on the specific nepovirus and the detection method used. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The near-unanimity-closed minions of Boolean functions, i.e., the clonoids whose target algebra contains a near-unanimity function, are completely described. The key concept towards this result is the minorant-minor partial order and its order ideals. [ABSTRACT FROM AUTHOR]

We describe the ordering of a class of clones by minion homomorphisms, also known as minor preserving maps or height 1 clone homomorphisms. The class consists of all clones on finite sets determined by binary relations whose projections to both coordinates have at most two elements. This class can be alternatively described up to minion homomorphisms as the class of multisorted Boolean clones determined by binary relations. We also introduce and apply the concept of a minion core which provides canonical representatives for equivalence classes of clones, more generally minions, on finite sets. [ABSTRACT FROM AUTHOR]

Abstract Background DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. Results We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12–15 h of sequencing. Conclusion Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

Accurate taxonomic species identification is essential to the study of mammals. Despite this necessity, rapid and accurate identification of cryptic, understudied, and elusive mammals remains challenging. Traditional barcoding of mitochondrial genes is standard for molecular identification but requires time-consuming wet-lab methodologies. Recent bioinformatic advancements for nanopore sequencing data offer exciting opportunities for noninvasive and field-based identification of mammals. Nanopore adaptive sampling (NAS), a polymerase chain reaction (PCR)-free method, selectively sequences regions of DNA according to user-specified reference databases. Here, we utilized NAS to enrich mammalian mitochondrial genome sequencing to identify species. Fecal DNA extractions were sequenced from 9 mammals, several collected in collaboration with Minnesota Tribal Nations, to demonstrate utility for NAS barcoding of noninvasive samples. By mapping to the entire National Center for Biotechnology Information mammalian mitochondrial reference genome database and bioinformatically analyzing highly similar matches, we successfully produced species identifications for all fecal samples. Eight of 9 species identifications matched previous PCR or animal/fecal appearance-based identifications. For the ninth species, our genetic data indicate a misidentification stemming from the original study. Our approach has a range of applications—particularly in field-based wildlife research, conservation, disease surveillance, and monitoring of wildlife trade. Of importance to Minnesota tribes is invasive species monitoring, detections, and confirmation as climate impacts cause changes in biodiversity and shifts in species distributions. The rapid assessment techniques described here will be useful as new introductions and range expansions of native and invasive species may first be detected by the presence of signs such as scat rather than direct observations and will be helpful for chronically understaffed tribal natural resources agencies. [ABSTRACT FROM AUTHOR]

Background: DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. Results: We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12–15 h of sequencing. Conclusion: Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications. [ABSTRACT FROM AUTHOR]

We developed a technology for detecting and quantifying trace nucleic acids using a bracketing protocol designed to yield a copy number with approximately ± 20% accuracy across all concentrations. The microRNAs (miRNAs) let-7b, miR-15b, miR-21, miR-375 and miR-141 were measured in serum and urine samples from healthy subjects and patients with breast, prostate or pancreatic cancer. Detection and quantification were amplification-free and enabled using osmium-tagged probes and MinION, a nanopore array detection device. Combined serum from healthy men (Sigma-Aldrich, St. Louis, MO, USA #H6914) was used as a reference. Total RNA isolated from biospecimens using commercial kits was used as the miRNA source. The unprecedented ± 20% accuracy led to the conclusion that miRNA copy numbers must be normalized to the same RNA content, which in turn illustrates (i) independence from age, sex and ethnicity, as well as (ii) equivalence between serum and urine. miR-21, miR-375 and miR-141 copies in cancers were 1.8-fold overexpressed, exhibited zero overlap with healthy samples and had a p-value of 1.6 × 10−22, tentatively validating each miRNA as a multi-cancer biomarker. miR-15b was confirmed to be cancer-independent, whereas let-7b appeared to be a cancer biomarker for prostate and breast cancer, but not for pancreatic cancer. [ABSTRACT FROM AUTHOR]

Influenza A virus poses significant public health challenges due to its high mutation rate and zoonotic potential. Whole-genome sequencing (WGS) is crucial for monitoring and characterizing these viruses. Oxford Nanopore Technologies (ONT) and Illumina next-generation sequencing platforms are commonly used, with ONT being advantageous for its long-read capabilities, portability, and unique ability to access raw data in real-time during sequencing, making it suitable for rapid outbreak responses. This study optimizes the ONT Ligation Sequencing Influenza A Whole Genome protocol by refining RT-PCR kits, primers, and purification methods, and evaluating automation for high-throughput processing. The alternative RT-PCR kits, combined with alternative primers, significantly improved read depth coverage and reduced short, untargeted reads compared to the original ONT protocol. The improvement was particularly evident in the minimum read depth coverage of polymerase segments, which often face challenges with achieving uniform coverage, displaying higher coverage at the 5’ and 3’ termini, and lower coverage in the central regions. This optimized protocol for targeted influenza A WGS not only enhances sequencing quality and efficiency, but is applicable to all NGS platforms, making it highly valuable for studying influenza adaptation and improving surveillance. Additionally, this protocol can be further refined and adapted for the sequencing of other pathogens, broadening its utility in various pathogen monitoring and response efforts.

BackgroundMitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods.ResultsWith the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80-90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes.ConclusionsThis method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Abstract Objectives Sabkhas represent polyextreme environments characterized by elevated salinity levels, intense ultraviolet (UV) radiation exposure, and extreme temperature fluctuations. In this study, we present the complete genomes of five bacterial isolates isolated from the sabkha-shore region and investigate their genomic organization and gene annotations. A better understanding of the bacterial genomic organization and genetic adaptations of these bacteria holds promise for engineering microbes with tailored functionalities for diverse industrial and agricultural applications, including bioremediation and promotion of plant growth under salinity stress conditions. Data description We present a comprehensive genome sequencing and annotation of five bacteria (kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11) obtained from the shores of the Abu Dhabi Sabkha region. Initial bacterial identification was conducted through 16 S rDNA amplification and sequencing. Employing a hybrid genome assembly technique combining Illumina short reads (NovaSeq 6000) and Oxford Nanopore long reads (MinION), we obtained complete annotated high-quality gap-free genome sequences. The genome sizes of the kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11 isolates were determined to be 2.4 Mb, 4.1 Mb, 2.9 Mb, 5.05 Mb, and 4.1 Mb, respectively. Our analysis conclusively assigned the bacterial isolates as Staphylococcus capitis (kcgeb_sa), Bacillus spizizenii (kcgeb_sc and kcgeb_S11), Pelagerythrobacter marensis (kcgeb_sd), and Priestia aryabhattai (kcgeb_S4).

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Healths capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia.

Objectives: Sabkhas represent polyextreme environments characterized by elevated salinity levels, intense ultraviolet (UV) radiation exposure, and extreme temperature fluctuations. In this study, we present the complete genomes of five bacterial isolates isolated from the sabkha-shore region and investigate their genomic organization and gene annotations. A better understanding of the bacterial genomic organization and genetic adaptations of these bacteria holds promise for engineering microbes with tailored functionalities for diverse industrial and agricultural applications, including bioremediation and promotion of plant growth under salinity stress conditions. Data description: We present a comprehensive genome sequencing and annotation of five bacteria (kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11) obtained from the shores of the Abu Dhabi Sabkha region. Initial bacterial identification was conducted through 16 S rDNA amplification and sequencing. Employing a hybrid genome assembly technique combining Illumina short reads (NovaSeq 6000) and Oxford Nanopore long reads (MinION), we obtained complete annotated high-quality gap-free genome sequences. The genome sizes of the kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11 isolates were determined to be 2.4 Mb, 4.1 Mb, 2.9 Mb, 5.05 Mb, and 4.1 Mb, respectively. Our analysis conclusively assigned the bacterial isolates as Staphylococcus capitis (kcgeb_sa), Bacillus spizizenii (kcgeb_sc and kcgeb_S11), Pelagerythrobacter marensis (kcgeb_sd), and Priestia aryabhattai (kcgeb_S4). [ABSTRACT FROM AUTHOR]

While originally intending to explore the venom gland microbiome of the desert hairy scorpion Hadrurus arizonensis Ewing, 1928, nanopore sequencing serendipitously recovered complete mitochondrial genomes for this iconic arachnid. Phylogenetic analysis of these high-quality genomes places Hadrurus as sister to Uroctonus, in agreement with some phylogenomic hypotheses. Additionally, we reveal significant genetic variation among individuals from the same population, highlighting the potential of mitogenomics for population genetics and phylogeography. This study showcases the effectiveness and affordability of nanopore sequencing for research with non-model organisms, opening new avenues for investigating arachnid biodiversity, evolution, and biogeography. [ABSTRACT FROM AUTHOR]

During the month of February in 2019, the Western USA experienced elevated precipitation levels, corresponding to atmospheric river events, ending a drought period. Rainwater samples were collected at four time points across two weeks and analyzed by microscopy, analytical chemistry, and long-read sequencing methods. Quantification of whole cells showed concentrations of > 106 cells/L. Imaged cells from fluorescent and scanning electron microscopy included microeukaryotes. Analytic chemistry detected Na+ and Cl− ions, which were in agreement with back trajectories of an oceanic origin and atmospheric river occurrence. Taxonomic investigation of long-read sequences generated from the Nanopore MinION resulted in a high proportion of read assignments to fungal groups. For bacterial taxonomies, common rainwater-associated bacterial genera were present at higher proportions than other bacterial groups: Erwinia, Hymenobacter, Pseudomonas, and Pantoea. The microscopy data support the potential of intact and viable cell wet deposition into local environments and the taxonomic identification of common atmospheric-associated bacterial genera from long-read sequencing highlights the potential usefulness of this platform for atmospheric samples and field campaigns. [ABSTRACT FROM AUTHOR]

Molecular diagnostics in combination with morphological identification is the method of choice for several cryptic microbial plant pathogens. For some diagnostic applications, traditional sequencing techniques can be time consuming, making them ill-suited for biosecurity incursion responses, where accurate results are needed in real time. More rapid next generation sequencing tools must be tested and compared with traditional methods to assess their utility in biosecurity applications. Here utilizing 95 samples infected with fungal pathogen Phyllosticta cavendishii, from a recent incursion in Australia, we compare species identification success using Internal Transcribed Spacer (ITS) gene barcode on conventional Sanger and Oxford Nanopore MinION sequencing platforms. For Sanger sequencing, the average pairwise identity percentage score between generated consensus sequences and P. cavendishii sequence from holotype material on NCBI database was 99.9% ± SE 0.0 whereas for MinION sequencing the average pairwise identity percentage was 99.1% ± SE 0.1. Relatively larger consensus sequences (mean 486 bp ± SE 2.4) were generated by Sanger sequencing compared to MinION sequencing (mean 435 bp ± SE 4.6). Our results confirm that both sequencing methods can reliably identify P. cavendishii. MinION sequencing, provided quicker results compared to Sanger sequencing and demonstrated diagnostic competence, with the added advantage of being portable, for front-line "point of incursion" biosecurity applications. [ABSTRACT FROM AUTHOR]

ABSTRACT Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.

Protist diversity studies are frequently conducted using DNA metabarcoding methods. Currently, most studies have utilized short read sequences to assess protist diversity. One limitation of using short read sequences is the low resolution of the markers. For better taxonomic resolution longer sequences of the 18S rDNA are required because the full‐length has both conserved and hypervariable regions. In this study, a new primer pair combination was used to amplify the full‐length 18S rDNA and its efficacy was validated with a test community and then validated with field samples. Full‐length sequences obtained with the Nanopore MinION for protist diversity from field samples were compared with Illumina MiSeq V4 and V8‐V9 short reads. Sequences generated from the high‐throughput sequencers are Amplicon Sequence Variants (ASVs). Metabarcoding results show high congruency among the long reads and short reads in taxonomic annotation at the major taxonomic group level; however, not all taxa could be successfully detected from sequences. Based on the criteria of ≥95% similarity and ≥1000 bp query length, 298 genera were identified by all markers in the field samples, 250 (84%) were detected by 18S, while only 226 (76%) by V4 and 213 (71%) by V8‐V9. Of the total 85 dinoflagellate genera observed, 19 genera were not defined by 18S dinoflagellate ASVs compared to only three among the total 52 diatom genera. The discrepancy in this resolution is due to the lack of taxonomically available 18S reference sequences in particular for dinoflagellates. Overall, this preliminary investigation demonstrates that application of the full‐length 18S rDNA approach can be successful in field studies. [ABSTRACT FROM AUTHOR]

Trade in pangolins is illegal, and yet tons of their scales and products are seized at various ports. These large seizures are challenging to process and comprehensively genotype for upstream provenance tracing and species identification for prosecution. We implemented a scalable DNA barcoding pipeline in which rapid DNA extraction and MinION sequencing were used to genotype a substantial proportion of pangolin scales subsampled from 2 record shipments seized in Singapore in 2019 (37.5 t). We used reference sequences to match the scales to phylogeographical regions of origin. In total, we identified 2346 cytochrome b (cytb) barcodes of white‐bellied (Phataginus tricuspis) (from 1091 scales), black‐bellied (Phataginus tetradactyla) (227 scales), and giant (Smutsia gigantea) (1028 scales) pangolins. Haplotype diversity was higher for P. tricuspis scales (121 haplotypes, 66 novel) than that for P. tetradactyla (22 haplotypes, 15 novel) and S. gigantea (25 haplotypes, 21 novel) scales. Of the novel haplotypes, 74.2% were likely from western and west‐central Africa, suggesting potential resurgence of poaching and newly exploited populations in these regions. Our results illustrate the utility of extensively subsampling large seizures and outline an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies. [ABSTRACT FROM AUTHOR]

In view of the current biodiversity crisis and our need to preserve and improve ecosystem functioning, efficient means for characterizing and monitoring biodiversity are required. DNA barcoding, especially when coupled with new sequencing technologies, is a promising method that can, in principle, also be employed by taxonomic lay people. In this study we compare the performance of DNA barcoding by means of a third-generation sequencing technology, nanopore sequencing with classical Sanger sequencing, based on a sample of invertebrates collected from moss pads in a bog in Austria. We find that our nanopore sequencing pipeline generates DNA barcodes that are at least as good as barcodes generated with Sanger sequencing, with the MinION producing better results than the Flongle flowcell. We further find that while many arthropod taxa are well covered in the international reference DNA barcode database BOLD, this clearly is not the case for important taxa like mites and springtails, which hampers large-scale biodiversity assessments. Based on examples from our study we further highlight which factors might be responsible for ambiguous species identification based on BOLD and how this can, at least partly, be solved. [ABSTRACT FROM AUTHOR]

We consider classes of Boolean functions stable under compositions both from the right and from the left with clones. Motivated by the question how many properties of Boolean functions can be defined by means of linear equations, we focus on stability under compositions with the clone of linear idempotent functions. It follows from a result by Sparks that there are countably many such linearly definable classes of Boolean functions. In this paper, we refine this result by completely describing these classes. This work is tightly related with the theory of function minors, stable classes, clonoids, and hereditary classes, topics that have been widely investigated in recent years by several authors including Maurice Pouzet and his coauthors. [ABSTRACT FROM AUTHOR]

Abundant host and bacterial sequences can obscure the detection of less prevalent viruses in untargeted next-generation sequencing (NGS). Efficient removal of these non-targeted sequences is vital for accurate viral detection. This study presents a novel 28S ribosomal RNA (rRNA) RT-qPCR assay designed to assess the efficiency of avian rRNA depletion before conducting costly NGS for the detection of avian RNA viruses. The comprehensive evaluation of this 28S-test focuses on substituting DNase I with alternative DNases in our established depletion protocols and finetuning essential parameters for reliable host rRNA depletion. To validate the effectiveness of the 28S-test, we compared its performance with NGS results obtained from both Illumina and Nanopore sequencing platforms. This evaluation utilized swab samples from chickens infected with highly pathogenic avian influenza virus, subjected to established and modified depletion protocols. Both methods significantly reduced host rRNA levels, but using the alternative DNase had superior performance. Additionally, utilizing the 28S-test, we explored cost- and time-effective strategies, such as reduced probe concentrations and other alternative DNase usage, assessed the impact of filtration pre-treatment, and evaluated various experimental parameters to further optimize the depletion protocol. Our findings underscore the value of the 28S-test in optimizing depletion methods for advancing improvements in avian disease research through NGS. [ABSTRACT FROM AUTHOR]

Apicomplexan haemoparasites generate significant morbidity and mortality in humans and other animals, particularly in many low‐to‐middle income countries. Malaria caused by Plasmodium remains responsible for some of the highest numbers of annual deaths of any human pathogen, whilst piroplasmids, such as Babesia and Theileria can have immense negative economic effects through livestock loss. Diagnosing haemoparasites via traditional methods like microscopy is challenging due to low‐level and transient parasitaemia. PCR‐based diagnostics overcome these limitations by being both highly sensitive and specific, but they may be unable to accurately detect coinfections or identify novel species. In contrast, next‐generation sequencing (NGS)‐based methods can characterize all pathogens from a group of interest concurrently, although, the short‐read platforms previously used have been limited in the taxonomic resolution achievable. Here, we used Oxford Nanopore Technologies' (ONT) long‐read MinION™ sequencer to conduct apicomplexan haemoparasite metabarcoding via sequencing the near full‐length 18S ribosomal RNA gene, demonstrating its ability to detect Babesia, Hepatozoon, Neospora, Plasmodium, Theileria and Toxoplasma species. This method was tested on blood‐extracted DNA from 100 dogs and the results benchmarked against qPCR and Illumina‐based metabarcoding. For two common haemoparasites, nanopore sequencing performed as well as qPCR (kappa agreement statistics > 0.98), whilst also detecting one pathogen, Hepatozoon felis, missed by the other techniques. The long‐reads obtained by nanopore sequencing provide an improved species‐level taxonomic resolution whilst the method's broad applicability mean it can be used to explore apicomplexan communities from diverse mammalian hosts, on a portable sequencer that easily permits adaptation to field use. [ABSTRACT FROM AUTHOR]

Cichlids are relevant to biological research for their craniofacial variations that are analogous to human structure and associated congenital anomalies. However, only a limited number of cichlids have genetic information available. Investigating cichlids and adding to the body of knowledge about them may provide better insights into studying developmental biology and craniofacial structure. The angelfish, Pterophyllum scalare, is one cichlid for which we lack genetic information including a draft transcriptome assembly.This work is the first to provide a draft transcriptome and annotation using long-read Nanopore sequencing for P. scalare. Total RNA was extracted from angelfish tissue, and a cDNA-PCR library was prepared. Sequencing was performed on a singular R.9.4.1 MinION flow cell for 84 h. Various bioinformatic tools were then employed to assemble the sequencing reads into a transcriptome. The transcriptome was then annotated against various databases.23 million sequencing reads were collected totalling 21.9 Gb. The N50 sequencing read length was 1255 bp and the mean read length was 938. The data had an initial mean Phred score of 10.04. After assembly, the final transcriptome consists of 98,125 transcripts with a mean length of 1552 and N50 length of 2277. The transcriptome has a completeness of 80.5% as assessed by BUSCO. Functional annotation revealed pathways related to signal transduction, carbohydrate metabolism, and transcription are the most annotated in the transcriptome.

The effects that mycobiomes have on physiological traits in turfgrasses are poorly understood. Drought tolerance, an economically and ecologically important trait, can be influenced by symbiotic fungi. In this two-year study, we evaluated the mycobiome associated with tall fescue exposed to prolonged periods of drought stress in a rainout shelter. Twelve plants, comprising six sets of half-sibs (progenies having one parent in common), one exhibiting a drought-tolerant phenotype and the other a drought-susceptible phenotype, were selected for analysis each year. The mycobiomes associated with the shoot, root, and rhizosphere soil were evaluated for each tall fescue half-sib pair using both short-read Illumina MiSeq and long-read Oxford Nanopore Technology (ONT) MinION sequencing pipelines. Both platforms sequenced portions of the fungal nuclear ribosomal RNA genes. The Illumina MiSeq sequenced the internal transcribed spacer region (ITS, 600 bp), while the ONT MinION covered the small subunit, ITS, and partial large subunit (4,600 bp). Both sequencing pipelines revealed that the mycobiomes associated with the roots, shoots, and soil were significantly different. The ONT MinION pipeline identified more diverse fungal lineages and had higher taxonomic resolution than the Illumina pipeline. Our results also indicated that root pathogens may play a more important role than endophytic or mycorrhizal symbionts in tall fescue drought stress tolerance.

Porcine circovirus type 2 (PCV2) is responsible for a number of porcine circovirus-associated diseases (PCVAD) that can severely impact domestic pig herds. For a non-enveloped virus with a small genome (1.7 kb ssDNA), PCV2 is remarkably diverse, with eight genotypes (a-h). New genotypes of PCV2 can spread through the migration of wild boar, which are thought to infect domestic pigs and spread further through the domestic pig trade. Despite a large swine population, the diversity of PCV2 genotypes in Ukraine has been under-sampled, with few PCV2 genome sequences reported in the past decade. To gain a deeper understanding of PCV2 genotype diversity in Ukraine, samples of blood serum were collected from wild boars (n = 107) that were hunted in Ukraine during the November-December 2012 hunting season. We found 34/107 (31.8%) prevalence of PCV2 by diagnostic PCR. For domestic pigs, liver samples (n = 16) were collected from a commercial market near Kharkiv in 2019, of which 6 out of 16 (37%) samples were positive for PCV2. We sequenced the genotyping locus ORF2, a gene encoding the PCV2 viral capsid (Cap), for 11 wild boar and six domestic pig samples in Ukraine using an Oxford Nanopore MinION device. Of 17 samples with resolved genotypes, the PCV2 genotype b was the most common in wild boar samples (10 out of 11, 91%), while the domestic pigs were infected with genotypes b and d. We also detected genotype b/d and b/a co-infections in wild boars and domestic pigs, respectively, and for the first time in Ukraine we detected genotype f in a wild boar from Poltava. Building a maximum-likelihood phylogeny, we identified a sublineage of PCV2 genotype b infections in both wild and domestic swine, suggesting a possible epizootic cluster and an ecological interaction between wild boar and domestic pig populations in northeastern Ukraine.

Abstract Background Early availability of pathogen identification in urinary tract infections (UTIs) has critical importance in disease management. Metagenomic next-generation sequencing (mNGS) has the potential to transform how acute and serious infections are diagnosed by offering unbiased and culture-free pathogen detection. However, clinical experience with application of the mNGS test is relatively limited. Methods We therefore established a MinION-based mNGS pathogens diagnostic platform and evaluated its potential for clinical implementation in UTIs with clinical samples. 213 urine samples from patients with suspected UTIs were included and subjected to mNGS testing using the MinION platform. mNGS results were compared to the gold standard of clinical culture and composite standard of combining clinical testing, confirmatory qPCR testing, and clinical adjudication by doctors. Results The mNGS exhibited a sensitivity of 81.4% and a specificity of 92.3%, along with a positive predictive value of 96.6%, a negative predictive value of 64.9%, and an overall accuracy of 84.4%, all of which were determined based on the gold standard of routine culture results. When assessed against the composite standard, the sensitivity and specificity both increased to 89.9% and 100%, respectively, while the accuracy rose to 92.4%. Notably, the positive predictive value and negative predictive value also saw improvements, reaching 100% and 76.8%, respectively. Moreover, this diagnostic platform successfully identified dsDNA viruses. Among the 65 culture-negative samples, the viral detection rate reached 33.8% (22/65) and was subsequently validated through qPCR. Furthermore, the automatic bioinformatics pipeline we developed enabled one-click analysis from data to results, leading to a significant reduction in diagnosis time. Conclusion These results demonstrate that the pathogen detection performance of mNGS is sufficient for diagnostic testing in clinical settings. As the method is generally unbiased, it can improve diagnostic testing of UTIs and other microbial infections.

Abstract Background Oxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT’s recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein. Results Average read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (

Balsa (Ochroma pyramidale) is fast-growing forest plant species introduced to Indonesia with limited genetic information. Genetic information can be obtained through molecular assessment which is now feasible due to sequencing technology development. This is supported by the third-generation sequencer technology, which has been developed using long-read sequencing technology. MinION Oxford Nanopore Technology is one of the long-read sequence-based sequencers with a real-time process and portable. This study aims to generate genomic data and analyze the phylogenetic relationship of balsa (O. pyramidale) based on long-read sequences with MinION Oxford Nanopore Technologies. Balsa long-read sequencing generated a partial chloroplast genome (cpDNA) sequence of 155,430 bp, which can be used for further DNA barcode-based phylogenetic analysis from the chloroplast genome. Phylogenetic analysis showed that the balsa species (O. pyramidale) was genetically grouped in one clade with other O. pyramidale species in phylogeny analysis based on rbcL, matK, and a combination of rbcL and matK genes indicated that those genes were a suitable marker for phylogenetic analysis in balsa species (O. pyramidale).

Abstract Background The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides. Results In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on–off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source. Conclusions Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito species-level identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications.

The Blastocystis subtype ST10 has been recognized to contain a great deal of diversity at the sequence level, potentially indicating the presence of multiple new STs within the clade. However, the data needed to validate these new STs were not available. To help resolve this diversity, full‐length small subunit (SSU) rRNA gene reference sequences were generated using Oxford Nanopore MinION long‐read sequencing from 21 samples representing multiple domestic and wild hosts and geographic regions and covering the sequence diversity previously described using fragments of the SSU rRNA gene. Phylogenetic and pairwise distance analyses were used to compare full‐length sequences of the SSU rRNA gene generated in this study with all other valid STs of Blastocystis. We present data supporting the division of ST10/ST23 cluster into five subtypes, ST10, ST23, and three new subtypes with the proposed ST designations of ST42, ST43, and ST44. As the host range of Blastocystis continues to expand with new subtypes and new hosts being frequently identified, the reference sequences provided in this study will assist in accurate sequence classification and help to clarify the epidemiology of this common intestinal microeukaryote. [ABSTRACT FROM AUTHOR]

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION's versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic. [ABSTRACT FROM AUTHOR]

Introduction: Whole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment. Methods: Herein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced. Results: After 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate. Discussion: The implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates. [ABSTRACT FROM AUTHOR]

Democratizing genomic data science, including bioinformatics, can diversify the STEM workforce and may, in turn, bring new perspectives into the space sciences. In this respect, the development of education and research programs that bridge genome science with "place" and world-views specific to a given region are valuable for Indigenous students and educators. Through a multi-institutional collaboration, we developed an ongoing education program and model that includes Illumina and Oxford Nanopore sequencing, free bioinformatic platforms, and teacher training workshops to address our research and education goals through a place-based science education lens. High school students and researchers cultivated, sequenced, assembled, and annotated the genomes of 13 bacteria from Mars analog sites with cultural relevance, 10 of which were novel species. Students, teachers, and community members assisted with the discovery of new, potentially chemolithotrophic bacteria relevant to astrobiology. This joint education-research program also led to the discovery of species from Mars analog sites capable of producing N-acyl homoserine lactones, which are quorum-sensing molecules used in bacterial communication. Whole genome sequencing was completed in high school classrooms, and connected students to funded space research, increased research output, and provided culturally relevant, place-based science education, with participants naming three novel species described here. Students at St. Andrew's School (Honolulu, Hawai'i) proposed the name Bradyrhizobium prioritasuperba for the type strain, BL16AT, of the new species (DSM 112479T = NCTC 14602T). The nonprofit organization Kauluakalana proposed the name Brenneria ulupoensis for the type strain, K61T, of the new species (DSM 116657T = LMG = 33184T), and Hawai'i Baptist Academy students proposed the name Paraflavitalea speifideiaquila for the type strain, BL16ET, of the new species (DSM 112478T = NCTC 14603T). [ABSTRACT FROM AUTHOR]

Streptococcus didelphis was once reported as related to severe infections in opossums. Thus, we present the first comprehensive whole-genome characterization of clinical S. didelphis strains isolated from white-eared opossums (Didelphis albiventris). Long-read whole-genome sequencing was performed using the MinION platform, which allowed the prediction of several genomic features. We observed that S. didelphis genomes harbor a cluster for streptolysin biosynthesis and a conserved genomic island with genes involved in transcriptional regulation (arlR) and transmembrane transport (bcrA). Antimicrobial resistance genes for several drug classes were found, including beta-lactam, which is the main antimicrobial class used in Streptococcus spp. infections; however, no phenotypical resistance was observed. In addition, we predicted the presence of 33 virulence factors in the analyzed genomes. High phylogenetic similarity was observed between clinical and reference strains, yet no clonality was suggested. We also proposed dnaN, gki, pros , and xpt as housekeeping candidates to be used in S. didelphis sequence typing. This is the first whole-genome characterization of S. didelphis , whose data provide important insights into its pathogenicity. [ABSTRACT FROM AUTHOR]

Several species of sacoglossan sea slugs possess the incredible ability to sequester chloroplasts from the algae they consume. These "photosynthetic animals" incorporate stolen chloroplasts, called kleptoplasts, into the epithelial cells of tubules that extend from their digestive tracts throughout their bodies. The mechanism by which these slugs maintain functioning kleptoplasts in the absence of an algal nuclear genome is unknown. Here, we report a draft genome of the sacoglossan slug Elysia crispata morphotype clarki, a morphotype native to the Florida Keys that can retain photosynthetically active kleptoplasts for several months without feeding. We used a combination of Oxford Nanopore Technologies long reads and Illumina short reads to produce a 786-Mb assembly (N50 = 0.459 Mb) containing 68,514 predicted protein-coding genes. A phylogenetic analysis found no evidence of horizontal acquisition of genes from algae. We performed gene family and gene expression analyses to identify E. crispata genes unique to kleptoplast-containing slugs that were more highly expressed in fed versus unfed developmental life stages. Consistent with analyses in other kleptoplastic slugs, our investigation suggests that genes encoding lectin carbohydrate-binding proteins and those involved in regulation of reactive oxygen species and immunity may play a role in kleptoplast retention. Lastly, we identified four polyketide synthase genes that could potentially encode proteins producing UV- and oxidation-blocking compounds in slug cell membranes. The genome of E. crispata is a quality resource that provides potential targets for functional analyses and enables further investigation into the evolution and mechanisms of kleptoplasty in animals. [ABSTRACT FROM AUTHOR]

Mycobacterium marinum (M. marinum) is a non-tuberculous mycobacterium (NTM) that can cause infectious diseases in aquatic animals and humans. Culture-based pathogen detection is the gold standard for diagnosing NTM infection. However, this method is time-consuming and has low positivity rates for fastidious organisms. Oxford Nanopore MinION sequencing is an emerging third-generation sequencing technology that can sequence DNA or RNA directly in a culture-independent manner and offers rapid microbial identification. Further benefits include low cost, short turnaround time, long read lengths, and small equipment size. Nanopore sequencing plays a crucial role in assessing drug resistance, clinical identification of microbes, and monitoring infectious diseases. Some reports on Mycobacterium tuberculosis (MTB) using nanopore sequencing have been published, however, there are few reports on NTM, such as M. marinum. Here, we report the use of nanopore sequencing for the diagnosis of M. marinum. [ABSTRACT FROM AUTHOR]

The marine tetraflagellate Cymbomonas tetramitiformis has drawn attention as an early diverging green alga that uses a phago-mixotrophic mode of nutrition (i.e. the ability to derive nourishment from both photosynthesis and bacterial prey). The Cymbomonas nuclear genome was sequenced previously, but due to the exclusive use of short-read (Illumina) data, the assembly suffered from missing a large proportion of the genome's repeat regions. For this study, we generated Oxford Nanopore long-read and additional short-read Illumina data and performed a hybrid assembly that significantly improved the total assembly size and contiguity. Numerous endogenous viral elements were identified in the repeat regions of the new assembly. These include the complete genome of a giant Algavirales virus along with many genomes of integrated Polinton-like viruses (PLVs) from two groups: Gezel-like PLVs and a novel group of prasinophyte-specific PLVs. The integrated ∼400 kb genome of the giant Algavirales virus is the first account of the association of the uncultured viral family AG_03 with green algae. The complete PLV genomes from C. tetramitiformis ranged between 15 and 25 kb in length and showed a diverse gene content. In addition, heliorhodopsin gene-containing repeat elements of putative mirusvirus origin were identified. These results illustrate past (and possibly ongoing) multiple alga–virus interactions that accompanied the genome evolution of C. tetramitiformis. [ABSTRACT FROM AUTHOR]

The blueberry industry, a fast-growing industry in Florida, is threatened by bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC). In 2016, distinct RSSC populations were found in Florida causing disease in blueberry. Currently, there are no tools that discriminate between RSSC populations pathogenic to blueberry and other RSSC strains. Early detection of specific RSSC strains is critical, and the Microbe Finder (MiFi) web application is a sensitive and specific tool used to detect known target pathogens in sequenced samples. MiFi is used to develop e-probes and subsequently query sample metagenomes with them. We report the development and validation of e-probes for three different RSSC populations pathogenic to blueberry using the MiFi platform. We also present preliminary data to assess the feasibility of a cost-effective integrated diagnostics system, MiFi, in combination with MinION, a pocket-sized, portable sequencer. The e-probes were validated in silico, generating simulated metagenomes; in vitro by using blueberry DNA spiked with pathogen DNA; and in vivo with soil and stem samples. We tested several methods of DNA extraction for soil and blueberry stems and several MinION libraries with or without barcodes. MiFi blueberry e-probes effectively identified target pathogens at a very specific level with high sensitivity. The MiFi system combined with MinION sequencing using Flongle cells was optimized to produce results in one day. Further optimization and tests can improve greatly the cost effectiveness and flexibility of this system by adding e-probes to test for new blueberry strains and many other pathogens affecting blueberry in one test. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Background Mitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods. Results With the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80–90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes. Conclusions This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

We report whole-genome sequencing of influenza A virus (IAV) with 100% diagnostic sensitivity and results available in 500× coverage from each of 16 reference virus isolates evaluated. Subgenomic viral sequences obtained in 3 cases using Sanger sequencing as the reference standard were identical to those obtained when sequenced using the MinION approach. An inter-laboratory comparison demonstrated reproducibility when comparing 2 independent laboratories at ≥99.8% across the entirety of the IAV genomes sequenced.

Abundant host and bacterial sequences can obscure the detection of less prevalent viruses in untargeted next-generation sequencing (NGS). Efficient removal of these non-targeted sequences is vital for accurate viral detection. This study presents a novel 28S ribosomal RNA (rRNA) RT-qPCR assay designed to assess the efficiency of avian rRNA depletion before conducting costly NGS for the detection of avian RNA viruses. The comprehensive evaluation of this 28S-test focuses on substituting DNase I with alternative DNases in our established depletion protocols and finetuning essential parameters for reliable host rRNA depletion. To validate the effectiveness of the 28S-test, we compared its performance with NGS results obtained from both Illumina and Nanopore sequencing platforms. This evaluation utilized swab samples from chickens infected with highly pathogenic avian influenza virus, subjected to established and modified depletion protocols. Both methods significantly reduced host rRNA levels, but using the alternative DNase had superior performance. Additionally, utilizing the 28S-test, we explored cost- and time-effective strategies, such as reduced probe concentrations and other alternative DNase usage, assessed the impact of filtration pre-treatment, and evaluated various experimental parameters to further optimize the depletion protocol. Our findings underscore the value of the 28S-test in optimizing depletion methods for advancing improvements in avian disease research through NGS.

IntroductionWhole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment.MethodsHerein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced.ResultsAfter 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate.DiscussionThe implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates.